Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDAC) is a cancer with an extremely low five-year survival rate, with most patients diagnosed with incurable metastatic disease. New therapies aimed at targeting the distinctive biology of PDAC cells are needed since current treatments offer little survival benefit. Therapies aimed at targeting cancer's unique metabolism have been successful in other cancers and can be applied to the treatment of PDAC. Complicating the study of metabolism in PDAC tumors is the fact that most of the tumor is composed of stromal cells such as fibroblasts, whereas little of the tumor is composed of cancer cells. To better understand PDAC metabolism and develop appropriate therapies, we must understand the metabolic differences between cancer and stromal cells. The main goal of this proposal is to investigate metabolic heterogeneity in pancreatic cancer. Since monolayer adherent cell culture systems have limitations in modeling heterogeneity, this proposal outlines a new approach for studying metabolism in sorted PDAC cell types in organoid co-cultures and mouse models. By studying 13C-labeled nutrient incorporation into stable macromolecules, we can bypass the problem posed by the short timescale of metabolic reactions. The goals of this project are to examine metabolic differences between PDAC cancer cells and fibroblasts in primary tumors (Aim 1) and compare the metabolism of these cells to that of tumor cells and fibroblasts within metastatic tumors (Aim 2). First, I will use 13C-nutrient tracing into stable macromolecules in organoid co-cultures and mouse models of PDAC to understand how nutrient use differs between PDAC cell types. I will then use Crispr-Cas9 to examine the requirement for different metabolic enzymes in cancer cells and stroma. Finally, I will use this method to explore differences in metabolism between cancer cells and fibroblasts within a primary to tumor to the metabolism of these cells in metastases in vivo. The proposed training plan will support me in my transition to independence. I have assembled a team of scientists with an outstanding track record of scientific and career mentoring to help me achieve my goal of becoming an independent scientist: Dr. Tyler Jacks, a leader in developing mouse models of cancer, Dr. David Tuveson, an expert in pancreatic cancer and stroma, and Dr. Brian Wolpin, a clinician with extensive expertise in pancreatic cancer treatment. These scientists will meet with me regularly as collaborators and members of my career mentoring committee. My training plan also outlines ways that I will cultivate scientific and career mentors, improve my science communication skills, develop teaching and mentoring skills, build my network, and learn lab management skills. Together, the scientific proposal and career development plan will give me the training and expertise I need to become a successful independent investigator in the field of cancer biology.